Thermostable polymerases have been isolated and purified from a number of eubacterial and archaeal (archaebacterial) sources. For example, Comb, et al. U.S. Pat. No. 5,352,778; (Oct. 4, 1994) describes a recombinant thermostable DNA polymerase from Thermococcus litoralis; Gelfand, et al. U.S. Pat. No. 5,079,352; (Jan. 7, 1992) discloses a thermostable enzyme purified from Thermus aquaticus; PCT WO92/09689 (Mathur, E. J.; published 11 Jun. 1992) describes a thermostable polymerase from Pyrococcus furiosus; PCT WO92/03556 (Gelfand, et al., published 5 Mar. 1992) describes a DNA polymerase from Thermotoga maritima; and WO92/06202 (Gelfand, et al.; published 16 Apr. 1992) describes a DNA polymerase from Thermosipho africanus.
Characteristics and properties such as optima of pH, temperature, degree of thermostability, and ionic requirements have been determined for many of these enzymes. Thermostable polymerases initially found great commercial appeal due to their ability to catalyze polymerization of DNA from a DNA template in the polymerase chain reaction (PCR). Other uses of thermostable polymerase include cycle sequencing of small amounts of DNA, sequencing of PCR products, standard sequencing reactions at elevated temperatures, and reverse transcription PCR type reactions. These techniques have been described in detail. (See, for example, G. A. Hawkins, et al., Biotechniques 16:418-20, 1994; M. A. Innis, et al., Proc. Natl. Acad. Sci. U.S.A. 85:9436-40, 1988; M. C. Kricker, et al., Gene 85:199-204, 1989; J. S. Lee, DNA Cell Biol. 10:67-73, 1991; T. W. Myers, et al., Biochemistry 30:7661-6, 1991; Wolfe, et al., Eur. J. Clin. Chem. Clin. Biochem. 30:717-27, 1992.)
Currently commercially available polymerases suffer from certain limitations when performing the above described procedures. DNA polymerases from Thermus aquaticus (Taq) and Thermus thermophilus (Tth) lack a sufficient proof-reading function as well as having reduced thermostability. Additionally, long PCR products are difficult to achieve when using Taq and Tth polymerases primarily because of the lack of proof-reading function. DNA prepared using these polymerases frequently requires modification before subsequent manipulations can be performed.
DNA polymerases such as those derived from Pyrococcus furiosus and Thermococcus litoralis have greater thermostability and exhibit a high degree of proof-reading capability. (See, for example, M. Bergseid, et al., Strategies in Molecular Biology 4(3):34-5, 1991; N. F. Cariello, et al., Nucleic Acids Res. 19:4193-8, 1991; K. A. Eckert, et al., PCR Methods and Applications 1(1):17-24, 1991; H. Kong, et al., J. Biol. Chem. 268:1965-75, 1993; T. A. Kunkel, et al., "Fidelity of DNA polymerase used in polymerase chain reactions," Current Communication in Molecular Biology, pp. 5-10, 1989; K. S. Lundberg, et al., Gene 108:1-6, 1991.) These polymerases, although improvements over the former polymerases, have several drawbacks. Two disadvantages of these polymerases are the higher incubation temperatures to which these polymerases are accustomed as well as an overactive 3' to 5' exonuclease function.
Therefore, it is desirable to develop a thermostable DNA polymerase which functions optimally at approximately 80.degree. C., has increased thermostability, and exhibits proof-reading capabilities that do not overwhelm polymerase function.